Regulation of enzyme function in vivo involves manifold control mechanisms, including those governing protein biosynthesis, transport, activation and catalytic efficiency. In the gram negative bacterium, E. coli, proteins localized between the cytoplasmic and outer membranes (the periplasmic space) are vectorially transported to this site as precursor forms of the mature protein. Proteolytic cleavage of an amino-terminal leader sequence of apparent hydrophobic character terminates the transport process. Related molecular changes, such as subunit assembly, cofactor and metal-ion binding and conformational alterations ultimately yield the mature enzyme. Alkaline phosphatase is one of the periplasmic proteins, a dimeric Zn(II) metalloenzyme. The stoichiometry of bound metal ion affects the expression of enzymatic activity as reflected in the equilibrium distribution of intermediates including the covalent phosphorylenzyme. These effects include the modulation of allosteric control, produced by subunit-subunit interactions, and detected as negative homotropic interactions affecting substrate and inhibitor binding. The objective of the proposed research is to elucidate the nature of these manifestations of enzymatic regulation by determining in detail the nature of molecular changes describing these phenomena. This includes studies of the isolated, purified precursor forms of the periplasmic proteins and the mechanism of their conversion as well as investigation of the consequences of effector binding to mature proteins such as alkaline phosphatase and the periplasmic-Pi binding protein. Differential scanning calorimetry is utilized as a primary tool in the investigation of structural changes in such events along with complementary instrumental methods.